Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor, as an Antitumor Drug Candidate in Mice.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Pharmacokinetic Characterization of LW6, a Novel Hypoxia-Inducible Factor-1α (HIF-1α) Inhibitor in Mice.

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUClast values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t1/2 > 1 h) and serum (t1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.

Application of a physiologically based pharmacokinetic model for the prediction of mirabegron plasma concentrations in a population with severe renal impairment.

We previously verified a physiologically based pharmacokinetic (PBPK) model for mirabegron in healthy subjects using the Simcyp Simulator by incorporating data on the inhibitory effect on cytochrome P450 (CYP) 2D6 and a multi-elimination pathway mediated by CYP3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7 and butyrylcholinesterase (BChE). The aim of this study was to use this PBPK model to assess the magnitude of drug-drug interactions (DDIs) in an elderly population with severe renal impairment (sRI), which has not been evaluated in clinical trials. We first determined the system parameters, and meta-analyses of literature data suggested that the abundance of UGT2B7 and the BChE activity in an elderly population with sRI was almost equivalent to and 20% lower than that in healthy young subjects, respectively. Other parameters, such as the CYP3A4 abundance, for an sRI population were used according to those built into the Simcyp Simulator. Second, we confirmed that the PBPK model reproduced the plasma concentration-time profile for mirabegron in an sRI population (simulated area under the plasma concentration-time curve (AUC) was within 1.5-times that of the observed value). Finally, we applied the PBPK model to simulate DDIs in an sRI population. The PBPK model predicted that the AUC for mirabegron with itraconazole (a CYP3A4 inhibitor) was 4.12-times that in healthy elderly subjects administered mirabegron alone, and predicted that the proportional change in AUC for desipramine (a CYP2D6 substrate) with mirabegron was greater than that in healthy subjects. In conclusion, the PBPK model was verified for the purpose of DDI assessment in an elderly population with sRI.

Determination of Mirabegron in rat plasma by UPLC-MS/MS after oral and intravenous administration

SUMMARY Mirabegron is a kind of β3 adrenergic receptor agonist which is an effective drug for the treatment of overactive bladder. In this research, a UPLC-MS/MS method is developed and validated for the study of mirabegron pharmacokinetic in rats. A protein precipitation method is applied for sample preparation with acetonitrile. m/z 397.3→379.6, m/z 326.4→121.0 for mirabegron, tolterodine (IS), respectively in the positive ion mode was performed for quantitation. The method is reliable and reproducible in our study (intra-day precision≤11.06%, inter-day precision≤11.43%) with concentration curves linear from 5 to 2500 ng/mL(R2>0.999). Stability studies demonstrated that mirabegron was stable under a variety of storage conditions. This method was successfully applied for determining mirabegron in rats after oral and intravenous administration.

RESUMO Mirabegron é um tipo de agonista do receptor adrenérgico beta 3 que demonstra eficácia no tratamento de bexiga hiperativa. Nesta pesquisa, o método UPLC-MS/MS é desenvolvido e validado para o estudo da farmacocinética mirabegron em ratos. Um método de precipitação de proteínas é aplicado para a preparação de amostras com acetonitrilo. 397.3 → 379.6 M / Z, M / Z 326.4 → 121.0 para mirabegron, tolterodina (IS), respectivamente, para o íon positivo foi realizado para quantificação. O método é fiável e reprodutível em nosso estudo (precisão intradia ≤ 11,06%; precisão entredia ≤ 11.43%), com curvas de concentração linear de 5 a 2 ng/ml (R2 > 0,999). Estudos de estabilidade demonstraram que mirabegron permanece estável sob uma variedade de condições de armazenamento. Este método foi aplicado com sucesso para a determinação de mirabegron em ratos após administração oral e intravenosa.

Sirolimus and mirabegron interaction in a hematopoietic cell transplant patient.

PURPOSE: Hematopoietic cell transplant patients are exposed to numerous classes of medications. Transplant practitioners must vigilantly monitor for drug interactions especially involving immunosuppressants. We report a hematopoietic cell transplant patient receiving sirolimus who developed supratherapeutic serum concentrations after initiating mirabegron. SUMMARY: A 31-year-old, 98 kg female received a second umbilical cord blood transplant four years after the first transplant for relapsed acute myeloid leukemia. Mycophenolate mofetil and sirolimus were utilized for graft versus host disease prophylaxis. The patient was receiving sirolimus 2 mg daily and the serum concentration on day 26 post-transplant (day + 26) was within therapeutic range (6.7 µg/L, goal range 3-12 µg/L). Her post-transplant course was complicated by BK viruria-associated cystitis for which she was started on mirabegron. Six days after starting the new medication (day + 33), the sirolimus serum concentration increased to 19.2 µg/L. Thus mirabegron was discontinued and sirolimus was held. Sirolimus was restarted once the serum concentration was within goal and subsequently stabilized with a combination of 1 mg and 2 mg daily for a total weekly dose of 10 mg. The proposed mechanisms of interaction include: (1) sirolimus inhibition of organic anion transporting polypeptide leading to increased mirabegron in the intestinal lumen; (2) mirabegron inhibition of P-glycoprotein leading to increased absorption of sirolimus and; (3) increased sirolimus absorption leading to increased sirolimus serum concentrations. CONCLUSION: To our knowledge, this is the first report of a potential drug interaction between sirolimus and mirabegron. Transplant specialists should be aware of this potential interaction when considering the concurrent use of these medications.

Phase I Population Pharmacokinetic Assessment of the Oral Bromodomain Inhibitor OTX015 in Patients with Haematologic Malignancies.

BACKGROUND AND OBJECTIVES: OTX015 (MK-8628) is a novel inhibitor of the bromodomain and extraterminal (BET)-bromodomain (BRD) protein family, binding specifically to bromodomains BRD2/3/4 and impacting the epigenetic regulation of several oncogenes. We characterized the pharmacokinetics of this first-in-class BET-BRD inhibitor administered as a single agent, including population pharmacokinetic modelling. METHODS: A dose-escalation, phase Ib study was performed with oral OTX015 in patients with haematologic malignancies, at doses starting from 10 mg once daily (QD) with continuous or discontinuous schedules. Five or eight blood samples were collected per patient for pharmacokinetic analysis. OTX015 plasma concentrations were determined using validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and analysed using a nonlinear mixed-effects modelling software program. A population pharmacokinetic model was fitted to the data, and patient demographics and clinical chemistry parameters were tested as predictive covariates on the model parameters. RESULTS: Blood samples were analysed from 81 patients treated with OTX015 at doses ranging from 10 to 160 mg QD or 40 mg twice daily (BID), and 633 time-plasma concentrations were available for analysis. A one-compartment open model with linear elimination adequately described OTX015 pharmacokinetics. The most significant covariate was lean body mass (LBM), which decreased the between-subject variability in apparent total body clearance (CL) and the volume of distribution (V). The estimated pharmacokinetic parameters were the absorption rate constant (k a) = 0.731 h(-1), V = 71.4 L and CL = 8.47 L·h(-1). CONCLUSION: The pharmacokinetics of oral OTX015 in patients with haematologic malignancies can be described with a one-compartment model. Population pharmacokinetic modelling of OTX015 plasma concentrations showed that LBM influences V and CL. These findings do not suggest the need for dose adjustment.

Pharmacological profile of the selective ß3-adrenoceptor agonist mirabegron in cynomolgus monkeys.

Mirabegron is a novel ß3-adrenoceptor agonist developed for the treatment of overactive bladder. To clarify the relationship between the pharmacological effects of mirabegron in monkeys and the clinical efficacy in patients with overactive bladder, the effect of mirabegron on bladder function was evaluated using cynomolgus monkeys. Quantitative PCR revealed that mRNA expression of ß3-adrenoceptors was most abundant (98 %) among ß-adrenoceptor subtypes in the bladder of cynomolgus monkeys. Mirabegron, which showed selective and potent agonistic activity on monkey ß3-adrenoceptors expressed in Chinese hamster ovary cells with EC50 value of 32 nmol/L and intrinsic activity of 0.8, induced concentration-dependent relaxation of bladder smooth muscle strips isolated from cynomolgus monkeys with EC50 values of 120 nmol/L in 20 mmol/L KCl stimulation and 43 nmol/L under 9.81 mN resting tension. In conscious cynomolgus monkeys, mirabegron decreased micturition frequency at oral doses of 1 and 3 mg/kg and increased mean volume voided per micturition at an oral dose of 3 mg/kg. Plasma concentration at which bladder function improved in the cynomolgus monkeys was similar to that at the clinically effective dose in patients with overactive bladder. These data suggest that the relaxant function in monkey bladder is mainly mediated by ß3-adrenoceptors similar to that in the human bladder and mirabegron showed efficacy on the bladder functions of the same parameters in clinical evaluation endpoints.

Hemoglobin adducts and urine metabolites of 4,4'-methylenedianiline after 4,4'-methylenediphenyl diisocyanate exposure of rats.

4,4'-Methylenediphenyl diisocyanate (MDI) is a very important component in the production of polyurethane. In a long-term experiment, designed to determine the carcinogenic and toxic effects of MDI, rats were exposed chronically for 3 and 12 months, to 0.0 (control), 0.26, 0.70 and 2.06 mg MDI/m3 as aerosols. Hemoglobin adducts and urine metabolites of MDI were determined at the different doses in order to develop methods to biomonitor workers exposed to MDI and to assess a risk resulting from such exposure. Hemoglobin adducts and urine metabolites of 4,4'-methylenedianiline (MDA) were found in all rats, including controls. MDA and N-acetyl-MDA (AcMDA) were quantified by GC-MS after derivatization with heptafluorobutyric anhydride. The dose-response relationships for hemoglobin adducts and urine metabolites were non-linear over this dose range. In urine, free AcMDA and MDA were found after base extraction. The amount of MDA present in urine and to a lesser extent the AcMDA found in urine correlate well with the corresponding amount determined as hemoglobin adducts for all dose groups. In order to release MDA from possible conjugates of MDA and AcMDA, urine was treated under strong acidic conditions. Following this procedure higher MDA levels were found than the sum of MDA and AcMDA from mild base hydrolysis. Similar results were obtained with the rats exposed for 3 and 12 months, indicating that a steady state had been reached by 3 months. In order to perform further investigations of the bronchoalveolar lavage fluid one group of animals was given a 1 week recovery period before sacrifice. Hemoglobin adducts from these animals showed a decrease of approximately 40% for all dose groups. According to the lifetime of rat erythrocytes the levels of hemoglobin adducts should have decreased by only 22%. This suggests that the erythrocytes with modified hemoglobin have a shorter lifespan. In order to exclude the possibility that hemoglobin adducts may have resulted from ingestion of hydrolyzed MDI via licking of the fur, a single dose experiment with rats exposed through the nose only or with the whole body was carried out. The only difference observed between these two exposure regimes was that the hemoglobin adduct levels of AcMDA after nose only exposure were significantly higher than after total body exposure. The presence of AcMDA in urine and as a hemoglobin adduct indicates that MDA was bioavailable after MDI exposure. The presence of MDA may contribute significantly to the carciongenic potential of MDI, since MDA has been shown to be carcinogenic in animals.

Propachlor-S-cysteine: a major circulating metabolite in the calf, pig and rat after administration of propachlor.

1. Propachlor-S-cysteine was the major metabolite found in systemic blood from rat, pig and calf given propachlor via the stomach. It was also the major metabolite found in the portal blood of pig; the portal blood of rat and calf was not examined. 2. Erythrocytes were the major transporter of propachlor metabolites in rat blood whereas plasma was the major transporter of these metabolites in pig and calf. 3. There was no evidence for metabolism of propachlor-S-cysteine by rat blood or by cytosol from rat, pig and calf erythrocytes.

Benzo(a)pyrene metabolism and plasma elimination rates of phenacetin, acetanilide and theophylline in man.

The plasma elimination rates of phenacetin, acetanilide and theophylline have been determined in 32 healthy subjects in an effort to find drugs resembling in their metabolism the carcinogen benzo(a)pyrene. The plasma half-lives and metabolic clearance rates of the three drugs were correlated with the inducibilities of aryl hydrocarbon hydroxylase (AHH) in mitogen-stimulated lymphocytes and the plasma half-lives and metabolic clearance rates of antipyrine determined in previous studies. Statistically significant correlations were found between the half-lives and metabolic clearance rates of phenacetin, acetanilide and theophylline and the AHH ratios except for the metabolic clearance rates of phenacetin which did not correlate. The correlations of the three drugs with the half-lives and metabolic clearance rates of antipyrine were equally good. Of all the drugs tested thus far for similarity in metabolism to benzo(a)pyrene, antipyrine showed the best association followed closely by theophylline.